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The Cdh5-CreERT2 transgene causes conditional Shb gene deletion in hematopoietic cells with consequences for immune

Qi He1, Xiujuan Li1,2, Kailash Singh1

  • 1Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden.

Scientific Reports
|May 19, 2019
PubMed

Insights

Conditional gene deletion in endothelial cells using Cdh5-CreERT2 may affect hematopoietic cells, confounding immune response studies. Bone marrow transplantation can mitigate these off-target effects, ensuring accurate endothelial-specific research.

Area of Science:

  • Immunology
  • Genetics
  • Cell Biology

Background:

  • The Cdh5-CreERT2 system is widely used for endothelial cell-specific gene deletion.
  • Understanding the role of the Shb gene in endothelial cells is crucial for melanoma immune responses.

Purpose of the Study:

  • To investigate the role of the endothelial cell Shb gene in B16F10 melanoma immune responses.
  • To evaluate potential confounding effects of Cdh5-CreERT2 in hematopoietic cells.

Main Methods:

  • Tamoxifen-induced conditional gene deletion in Cdh5-CreERT2/WT and Cdh5-CreERT2/Shbflox/flox mice.
  • Subcutaneous B16F10 melanoma cell injections.
  • Bone marrow transplantation experiments.
  • Analysis of Shb mRNA in myeloid cells and CD4+/FoxP3+ regulatory T cells (Tregs).
  • Reporter gene analysis (ROSA26-mTmG) to assess Cre activity in hematopoietic populations.

Main Results:

  • Tamoxifen treatment led to decreased Shb mRNA in tumor myeloid cells of Cdh5-CreERT2/Shbflox/flox mice, an effect abolished by bone marrow transplantation.
  • Differences in CD4+/FoxP3+ Tregs were also normalized after bone marrow transplantation.
  • Cdh5-CreERT2 activity was detected in bone marrow and spleen, indicating potential off-target effects in hematopoietic cells.
  • Spleen floxing was undetectable after two months, suggesting Cdh5-CreERT2 acts on non-renewable spleen hematopoietic cells.

Conclusions:

  • Conditional gene deletion in hematopoietic cells can confound studies aiming to assess endothelial-specific effects.
  • A preceding bone marrow transplantation strategy is recommended to achieve a reliable endothelial-specific phenotype.
  • Researchers should exercise caution when interpreting results from Cdh5-CreERT2-mediated gene deletion experiments.

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